加味丹参饮含药血清预处理对缺氧/复氧H9C2心肌细胞自噬相关基因Atg5和Beclin1 mRNA表达的影响
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摘要
目的探讨加味丹参饮含药血清预处理对缺氧/复氧H9C2心肌细胞自噬及其相关基因Atg5、Beclin1 mRNA表达的影响。方法将体外培养的H9C2心肌细胞随机分为空白血清组(CG)、缺氧/复氧组(HRG)、加味丹参饮含药血清组(JDG)、JDG+自噬抑制剂(3-MA)组(JIG)、JDG+自噬激动剂(RAPA)组(JAG)。倒置显微镜观察各组心肌细胞生长及形态变化;MTT比色法测定心肌细胞存活率;透射电子显微镜观察心肌细胞自噬体形态及数量;实时荧光定量PCR检测自噬相关基因Atg5、Beclin1 mRNA表达。结果与CG比较,HRG镜下心肌细胞变性坏死程度增加,细胞存活率下降(P<0.01),电镜下自噬体增多,实时荧光定量PCR示Atg5、Beclin1 mRNA表达上调;与HRG相比,JDG及JAG镜下心肌细胞变性坏死程度减轻,细胞存活率显著升高(P<0.01),电镜下自噬体增加,Atg5、Beclin1 mRNA表达进一步上调(P<0.05)。结论加味丹参饮预处理通过上调自噬相关基因Atg5、Beclin1 mRNA表达,增强细胞自噬,保护缺氧/复氧损伤H9C2心肌细胞。
Objective To investigate the influence of preconditioning with Jiawei Danshen decoction on expression level of autophagy related gene Atg5 and Beclin1 in H9C2 cardiomyocytes during hypoxia/reoxygenation injury.Methods H9C2 cardiomyocytes cultured in vitro were randomly divided into control group( CG),hypoxia/reoxygenation group( HRG),Jiawei Danshen decoction group( JDG),JDG + 3-methyladenine( a inhibitor of autophagy) group( JIG) and JDG +Rapamycin( RAPA)( an agonists of autophagy) group( JAG). The inverted microscope was used to observe cell growth and morphological changes.The cell viability of H9C2 cardiomyocytes were measured by MTT assay.Transmission electron microscope was applied to observe the changes of autophagosome formation in cardiomyocytes.The expression levels of Atg5 and Beclin1 mRNA were determined by qRT-PCR.Results Compared with those of CG,the degree of cell necrosis and damage were rised in HRG,the cell viability was declined( P<0.01),the autophagosome formation in cardiomyocytes was increased.Meanwhile,the expression levels of Atg5 and Beclin1 gene were up-regulated compared with those of HRG,the cardiomyocyte morphological structure of JDG and JAG were improved,the cell viability of JDG and JAG were increased significantly( P<0.01),the autophagosome formation in cardiomyocytes were increased.In addition,the expression levels of Atg5 and Beclin1 gene were up-regulated( P < 0. 05). Conclusions Preconditioning with Jiawei Danshen decoction has a protective effect on H9C2 cardiomyocytes during hypoxia/reoxygenation injury by up-regulating the expression levels of Atg5 and Beclin1 gene.
Objective To investigate the influence of preconditioning with Jiawei Danshen decoction on expression level of autophagy related gene Atg5 and Beclin1 in H9C2 cardiomyocytes during hypoxia/reoxygenation injury.Methods H9C2 cardiomyocytes cultured in vitro were randomly divided into control group( CG),hypoxia/reoxygenation group( HRG),Jiawei Danshen decoction group( JDG),JDG + 3-methyladenine( a inhibitor of autophagy) group( JIG) and JDG +Rapamycin( RAPA)( an agonists of autophagy) group( JAG). The inverted microscope was used to observe cell growth and morphological changes.The cell viability of H9C2 cardiomyocytes were measured by MTT assay.Transmission electron microscope was applied to observe the changes of autophagosome formation in cardiomyocytes.The expression levels of Atg5 and Beclin1 mRNA were determined by qRT-PCR.Results Compared with those of CG,the degree of cell necrosis and damage were rised in HRG,the cell viability was declined( P<0.01),the autophagosome formation in cardiomyocytes was increased.Meanwhile,the expression levels of Atg5 and Beclin1 gene were up-regulated compared with those of HRG,the cardiomyocyte morphological structure of JDG and JAG were improved,the cell viability of JDG and JAG were increased significantly( P<0.01),the autophagosome formation in cardiomyocytes were increased.In addition,the expression levels of Atg5 and Beclin1 gene were up-regulated( P < 0. 05). Conclusions Preconditioning with Jiawei Danshen decoction has a protective effect on H9C2 cardiomyocytes during hypoxia/reoxygenation injury by up-regulating the expression levels of Atg5 and Beclin1 gene.
引文
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10 Decker RS,Wildenthal K.Lysosomal alteration in hypoxic and reoxygenated hearts[J].Am J Pathol,1980;2(98):425-44.
11 Dosenko VE,Nagibin VS,Tumanovska LV,et al.Protective effect of autophagy in anoxia-reoxygenation of isolated cardiomyocyte[J].Autophagy,2006;2:305-6.
12 Boya P,Gonzlez-Polo RA,Casares N,et al.Inhibition of macroautophagy triggers apoptosis[J].Mol Cell Biol,2005;25(3):1025-40.
13史载祥.冠心病血运重建后中西医结合治疗思考[J].医学研究杂志,2007;36(7):2-5.
14刘杰民,纪云西,蒋历,等.细胞自噬是探索中医药微观机制的新思路[J].时珍国医国药,2013;24(2):425-6.
15李欣志,韩笑,秦思,等.心脏“阴阳自和”与心肌细胞自噬[J].中国中医基础医学杂志,2008;14(5):339-40.
2黄政德,王庆高,刘东亮,等.加味丹参饮预处理对心肌细胞内钙超载的延迟保护作用[J].湖南中医药大学学报,2007;27(2):37-9.
3李鑫辉,黄政德,喻嵘,等.加味丹参饮对血瘀证家兔缺血再灌注损伤心肌细胞凋亡影响[J].湖南中医药大学学报,2009;29(4):21-3.
4黄政德,葛金文,张玉生.加味丹参饮对家兔缺血再灌注损伤心肌延迟保护作用的研究[J].湖南中医杂志,2003;19(6):52-3.
5金泉克,肖海娟,孙钰,等.中药复方肠胃清含药血清对胃癌SGC7901细胞凋亡的影响[J].中国中医药信息杂志,2012;19(5):32-5.
6徐叔云,卞如濂.药理实验方法学[M].第2版.北京:人民卫生出版社,2002:45.
7王庆高,黄政德,肖健,等.加味丹参饮预处理对乳鼠缺氧/复氧心肌细胞的延迟保护作用及对蛋白激酶C的影响[J].中西医结合心脑血管病杂志,2007;5(10):953-5.
8 Sciarretta S,Volpe M,Sadoshima J.Mammalian target of rapamycin signaling in cardiac physiology and disease[J].Circ Res,2014;114(3):549-64.
9许荣,王宁,王晓明.自噬在心肌缺血再灌注损伤中的研究进展[J].中华老年心脑血管病杂志,2015;17(12):1327-8.
10 Decker RS,Wildenthal K.Lysosomal alteration in hypoxic and reoxygenated hearts[J].Am J Pathol,1980;2(98):425-44.
11 Dosenko VE,Nagibin VS,Tumanovska LV,et al.Protective effect of autophagy in anoxia-reoxygenation of isolated cardiomyocyte[J].Autophagy,2006;2:305-6.
12 Boya P,Gonzlez-Polo RA,Casares N,et al.Inhibition of macroautophagy triggers apoptosis[J].Mol Cell Biol,2005;25(3):1025-40.
13史载祥.冠心病血运重建后中西医结合治疗思考[J].医学研究杂志,2007;36(7):2-5.
14刘杰民,纪云西,蒋历,等.细胞自噬是探索中医药微观机制的新思路[J].时珍国医国药,2013;24(2):425-6.
15李欣志,韩笑,秦思,等.心脏“阴阳自和”与心肌细胞自噬[J].中国中医基础医学杂志,2008;14(5):339-40.